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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3792-3797
By
From the Department of Haematology and the University
Department of Clinical Biochemistry, Addenbrooke's NHS Trust,
Cambridge, UK.
Plasma and platelet factor Va represent different substrates for
activated protein C (APC). In this study, we have measured platelet-dependent APC resistance and the effect of aspirin and a
platelet glycoprotein IIbIIIa antagonist (GR144053F) on this phenomenon. In platelet rich plasma (PRP), progressive APC resistance was observed with increasing platelet activation. APC sensitivity ratios of 1.8, 1.7, and 1.4 were observed after platelet activation with thrombin receptor activating peptide (TRAP), collagen, and A23187,
respectively. Ultracentrifugation at 77,000g for 1 hour abolished APC resistance indicating that the phenotype is associated exclusively with the platelet membrane. APC resistance was not observed
in the presence of phosphatidylcholine-phosphatidylserine (PCPS)
vesicles or purified human plasma lipoproteins. APC resistance was
observed in the presence of platelet-derived microparticles, but to a
lesser degree than that in the presence of activated platelets. The
platelet-dependent APC resistance phenotype was also observed when
endogenous APC was generated by Protac (American Diagnostica,
Inc, Greenwich, CT). In vitro inhibition of platelet activation with aspirin had no effect, but the fibrinogen receptor antagonist, GR144053F, inhibited platelet-dependent APC resistance. These results indicate that platelet activation results in an APC-resistant phenotype comparable to that observed in the plasma of
patients with factor V gene mutations affecting critical APC cleavage
sites. This suggests that platelet activation at the site of
endothelial damage downregulates a critical natural anticoagulant mechanism. The antithrombotic effect of aspirin may be due to an
indirect effect on platelet-dependent APC resistance with reduced platelet retention within a developing thrombus. The more potent antithrombotic effect of glycoprotein IIbIIIa antagonists may in
addition be the result of reduced platelet factor Va expression and
modulation of the platelet-dependent APC resistance phenotype.
RESISTANCE TO activated protein C (APC
resistance) was described by Dahlback et al in 1993.1 The
molecular defect responsible for this phenomenon is typically a
mutation in the factor V gene, which results in a protein with an
altered amino acid sequence at a critical APC cleavage site. In almost
all cases, this is a point mutation within codon 506 (G1691A), which
alters the sequence of the initial APC cleavage site in the heavy chain of factor Va (factor V Leiden, FVR506Q).2-5 However, a
point mutation within codon 306 (G1091C) also causes an APC resistance phenotype. This latter mutation alters the APC cleavage site primarily responsible for loss of cofactor activity (factor V Cambridge, FVR306T).6 Resistance to APC and the FVR506Q mutation are
the most common identifiable defects in patients with venous
thromboembolism.7,8 In contrast, the prevalence of the
FVR506Q mutation is not increased in unselected patients with
myocardial infarction or thrombotic stroke.9 However,
recent evidence indicates that cerebrovascular disease may be
associated with APC resistance in the absence of the FVR506Q
mutation.10 This pattern of APC resistance may be an
acquired phenotype due to platelet activation and the presence of
platelet-derived microparticles in plasma samples. In vitro studies
have already shown an APC-resistant phenotype in the presence of
freeze-fractured platelets11-13 and Camire et
al14 have shown that platelet-derived factor Va cofactor
activity is sustained on the surface of activated platelets despite the
presence of APC.14
Characterization of a platelet-dependent APC resistance phenotype would
define a new mechanism of platelet-dependent thrombin generation and
further explain the role of platelets in arterial thrombosis. In
addition, it might provide insight into the antithrombotic mechanism of
established antiplatelet drugs, such as aspirin, and provide a target
mechanism for new antithrombotic agents. The purpose of this current
study was to quantify the effect of platelets on APC sensitivity and to
determine if platelet activation causes an APC resistance phenotype
comparable to that produced by mutations in the factor V gene. In
addition, we aimed to determine if the platelet-dependent APC
resistance phenotype could be mimicked by phospholipids, which were not
of platelet origin, for example atherogenic lipoproteins, which are
capable of supporting thrombin generation. Finally, we examined the
differential effect of aspirin and a platelet fibrinogen receptor
antagonist to determine if the antithrombotic effect of antiplatelet
therapy is due to a direct effect on platelet factor V expression or an
indirect effect due to reduced platelet localization within a
developing clot or thrombus.
Materials and Reagents
Preparation of Platelet-Poor Plasma (PPP), Platelet-Rich Plasma
(PRP), and Platelet Microparticle-Free Plasma
Freeze-Fractured Platelets Freeze-fractured PRP was prepared from pooled PRP after adjustment of platelet count to 400 × 109/L. The PRP was subjected to three cycles of freezing at 80°C and thawing at 37°C.
The freeze-fractured platelet suspension was double diluted in platelet
microparticle-free plasma, produced by ultracentrifugation at
77,000g for 1 hour, and stored at 80°C.
PCPS Vesicles Lyophilized phosphatidylserine:phosphatidylcholine vesicles (25%:75%) were reconstituted with deionized water and resuspended in microparticle-free plasma at a stock concentration of 1.5 mmol/L. This stock concentration was double diluted in microparticle-free plasma to obtain working concentrations of vesicles (1 to 500 µmol/L).Plasma Lipoproteins Single classes of human plasma lipoproteins (high-density lipoprotein [HDL], very low-density lipoprotein [VLDL], low-density lipoprotein [LDL], and oxidized LDL) were prepared as previously described.15 The protein content of each lipoprotein fraction was measured by a modified Lowry method and concentration expressed as protein content.16 The ability of lipoproteins to support thrombin generation in a purified component assay was performed as previously described.15Detection of Platelet-Derived Microparticles by ELISA Preparation of platelet microparticles. PRP was prepared as described above and applied to a C-series 16/40 gel-chromatography column packed with Sepharose CL-6B.17 The eluting solution used for gel filtration was phosphate-buffered saline (PBS), pH 7.2. Eluted gel-filtered platelets were counted on a Coulter STKS and adjusted to 150 × 109/L in PBS. For the purpose of generating a standard curve for the microparticle ELISA, microparticle formation was induced by activating gel filtered platelets with 1.0 mmol/L A23187 and 0.5 mmol/L CaCl2 (final concentrations). The suspensions were then centrifuged at 6,000g for 5 minutes to remove remnant platelets. The microparticle suspension was double diluted in PBS and 250 µL of each dilution was added to 750 µL of ultracentrifuged platelet microparticle-free plasma. Plate preparation. One hundred microliters of PRP and 100 µL of carbonate buffer (0.05 mol/L sodium carbonate-bicarbonate buffer, pH 9.6), were added to each well of a polystyrene microtiter plate. Plates were centrifuged at 1,000g for 12 minutes to produce a platelet monolayer coating. The supernatant was gently decanted, and 300 µL of the 1% formaldehyde and 0.01% human serum albumin fixative solution was added to each well and incubated for 1 hour. The plate was washed four times with platelet wash buffer (0.35% albumin Tyrode's solution containing 0.5 mmol/L prostaglandin E1 and heparin 5 U/mL, pH 7.4). Sample preparation. Platelets were removed from plasma samples by centrifugation at 6,000g for 5 minutes. The resultant PPP (containing microparticles) was added to 1% formaldehyde and human serum albumin fixative solution at a ratio of 1:2. This fixed solution was incubated with CD61 (DAKO), final concentration 1:250, for 2 hours at room temperature, resulting in antibody-microparticle complexes and excess free antibody. A total of 100 µL of this solution was incubated in wells in the platelet-coated microtiter plates for 2 hours at room temperature to capture free CD61. After a gentle wash cycle to remove microparticle complexed CD61, 100 µL of horseradish peroxidase-conjugated rabbit anti-mouse antibody (DAKO) at a concentration of 1:500 in platelet wash buffer was added to the plate and incubated for 1.5 hours at room temperature. The plate was then developed using 100 µL of 30 mg o-phenylenediamine dihydrochloride dissolved in 10 mL of citrate phosphate buffer (0.1 mol/L, pH 6.0) that had been activated by 20 µL H2O2. The development was stopped after 5 minutes by the addition of 150 µL 1 mol/L H2SO4. Absorbance was read at a wavelength of 492 nm. The amount of free CD61 detected was inversely proportional to the number of microparticles in the plasma sample. APC Sensitivity Ratios Activated partial thromboplastin times (APTT) were measured with COATEST APC Resistance-C kits on a KC10 coagulometer (Amelung, Germany). A total of 100 µL of sample was incubated with 100 µL of APTT reagent (purified phospholipid with colloidal silica as contact activator) for 5 minutes at 37°C. Clot formation was initiated by the addition of 100 µL of either CaCl2 (0.025 mol/L in Tris buffer containing 0.5% albumin) or CaCl2 plus APC (60 nmol/L). The APC sensitivity ratio was calculated as the APTT with APC divided by the APTT without APC. APC resistance is indicated by a ratio of less than 2.2.13Whole-Blood Dilute Thromboplastin Assay Nonanticoagulated venous whole blood was obtained from healthy volunteers. The initial 3 mL of blood was discarded, and the next 1 mL was used for the assay within 2 minutes of obtaining the sample. A total of 100 µL of this sample was added to 100 µL of thromboplastin diluted 1/1,000 in imidazole buffer and 100 µL of either CaCl2 or APC + CaCl2 from the COATEST kit. The mixture was stirred at 37°C in the KC10 coagulometer and clot times measured. Volunteers were then given 600 mg of aspirin orally, and a further blood sample was obtained 1.5 hours later. The whole blood dilute thromboplastin time was measured and platelet aggregation was performed to confirm that aspirin ingestion had resulted in at least a 50% reduction of collagen-induced platelet aggregation (100 µg/mL collagen final concentration).
Effect of Freeze-Fractured Platelets and Phospholipid Vesicles on APC Sensitivity The platelet count of PRP was varied and the APTT, with and without APC, was measured before and after freeze fracturing. The APTT of fresh PRP did not vary with platelet count and was the same as that of PPP. In contrast, in the presence of freeze-fractured PRP, there was a progressive increase in APC resistance with increasing platelet count. In the absence of APC, the APTT was constant, but in the presence of APC, the APTT was progressively reduced in relation to platelet count (Fig 1A). To determine if the APC resistance was associated with the platelet membrane or was due to a soluble factor released during platelet disruption, samples of PRP were ultracentrifuged. After ultracentrifugation at 77,000g for 1 hour, the APC resistance phenotype was abolished. The APTT of the ultracentrifuged PRP (154 seconds) was longer than that of the untreated PPP (120 seconds). However, platelet microparticles were detectable in PPP samples by ELISA and not in the ultracentrifuged PRP. When PPP samples were also ultracentrifuged, microparticles were no longer detectable and the APTT was the same as that of the ultracentrifuged PRP. These results indicate that the platelet-dependent APC resistance phenotype is associated exclusively with a particulate platelet fraction rather than a soluble factor released during platelet disruption. The prolongation of the APTT after ultracentrifugation of samples indicates that normal plasma typically contains some of the platelet-derived fraction that is responsible for APC resistance.
Effect of Atherogenic Lipoproteins on APC Resistance Purified lipoproteins (HDL, VLDL, LDL, and oxidized LDL) were added to microparticle free plasma and APC sensitivity ratios were determined. Maximum final concentrations in the APC resistance assay were 3,700 µg/mL for HDL, 2,400 µg/mL for VLDL, 1,000 µg/mL for LDL, and 620 µg/mL for oxidized LDL. These concentrations of VLDL and oxidized LDL were shown to support thrombin generation in the purified component thrombin generation assay, but did not affect APC sensitivity in the standard APC resistance assay. The result was therefore similar to that with PCPS vesicles.Quantification of APC Resistance Due to Platelet Activation The effect of platelet activation on APC sensitivity was determined after activation of platelets with TRAP, collagen, and the calcium ionophore A23187 (Fig 1B through D). Progressive APC resistance was observed at a TRAP concentration of 35 µmol/L and above, at a collagen concentration of 32 µg/mL and above, and an ionophore concentration of 6.25 µmol/L and above. The experiments were repeated using Protac to generate endogenous protein C activation within the plasma sample. The results were the same as that observed with addition of APC, with an identical pattern and degree of APC resistance with increasing platelet activation.Quantification of APC Resistance Due to Platelet-Derived Microparticles To determine if platelet-dependent APC resistance was associated with platelet-derived microparticles, as well as activated platelets, ultracentrifuged plasma was spiked with microparticles generated from gel-filtered platelets. The APTT of ultracentrifuged microparticle free plasma in the presence of APC was 165 seconds with an APC sensitivity ratio of 3.7. After addition of microparticles (produced from 300 × 109/L gel-filtered platelets with 1 µmol/L ionophore), this fell to 114 seconds with a ratio of 2.5. The plasma was then ultracentrifuged again at 77,000g for 1 hour. This removed all detectable microparticles and the APC resistance phenotype was abolished with the APTT returning to 165 seconds.Effect of In Vitro Inhibition of Platelet Activation Collagen-induced platelet activation was performed before and after incubation of PRP (platelet count 300 × 109/L) with aspirin and the platelet glycoprotein IIbIIIa antagonist GR144053F. Aspirin at a concentration of 40 µmol/L abolished collagen-induced platelet aggregation, but had no effect on the APTT in the presence of APC (F = 1.155, P = .288, analysis of variance [ANOVA] pairwise comparison Bonferroni test). GR144053F at a concentration of 65 nmol/L abolished collagen-induced platelet aggregation and prolonged the APTT in the presence of APC (F = 25.743, P < .001, ANOVA pairwise comparison Bonferroni test) (Fig 2).
Effect of In Vivo Inhibition of Platelet Activation With Aspirin To determine if aspirin influenced the effect of APC on thrombin generation in vivo, a dilute thromboplastin time was measured on nonanticoagulated fresh whole blood taken from healthy volunteers before and 90 minutes after ingestion of 600 mg of aspirin. In nonanticoagulated blood, there is a slow intrinsic activation of coagulation resulting in clot formation at variable times between 15 and 20 minutes. To minimize this activation, the blood sample was taken without a tourniquet and the first 3 mL of blood was discarded. When thromboplastin at a final dilution of 1 in 1,000 was added to whole blood, the variance of the clot time was eliminated and coagulation occurred within 6 minutes with synchronization between experiments, although clot times varied with different dilutions of thromboplastin. The same clot times were observed in plasma samples when the dilute thromboplastin was added at any time up to 5 minutes after collection of the blood sample.
This study has produced several new observations. First, platelet-dependent APC resistance results from activation of platelets with a variety of platelet agonists. The responses to TRAP and collagen suggest that platelet activation in vivo is likely to produce APC resistance. Second, the degree of APC resistance resulting from platelet activation is comparable to that observed in the plasma of patients with factor V gene mutations affecting critical APC cleavage sites. This indicates that platelet-dependent thrombin generation is not susceptible to regulation by the protein C system in the same way that thrombin generation is regulated in a platelet poor environment. Third, the ultracentrifugation studies indicate that platelet-dependent APC resistance is associated exclusively with the platelet membrane. Fourth, the phenomenon is not simply due to exposure of anionic phospholipid, as APC resistance was not induced by the addition of PCPS vesicles. Fifth, while atherogenic lipoproteins supported assembly of the prothrombinase complex, they did not confer APC resistance in the APC sensitivity assay. Therefore, lipoprotein-supported thrombin generation appears to be susceptible to regulation by the protein C system. Sixth, platelet-derived microparticles confer APC resistance, although to a lesser degree than that observed on the surface of activated platelets. Seventh, inhibition of platelet activation with a fibrinogen receptor antagonist attenuated APC resistance associated with platelet activation. This indicates a direct effect of this inhibitor on platelet-dependent APC resistance. In contrast, aspirin had no effect, suggesting that any modification of platelet-dependent APC resistance by aspirin is an indirect effect due to reduced platelet localization within a developing clot or thrombus.
We are grateful to Alan Giles, Mike Nesheim, and John Samis of Queens University, Kingston, Ontario, for much helpful discussion and the kind gift of PCPS vesicles.
Submitted September 15, 1998; accepted January 20, 1999.
Supported in part by grants from the Addenbrooke's Trust Endowment, the MRC, Anglia and Oxford Health Authority, and the Royal Society. C.D.B. is an MRC Clinician Scientist Fellow.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
Address reprint requests to Trevor Baglin, FRCP, Department of Haematology, Box 234, Addenbrooke's NHS Trust, Cambridge CB2 2QQ, UK; e-mail: tpb20{at}cus.cam.ac.uk.
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  R. J. S. Preston, S. Tran, J. A. Johnson, F. N. Ainle, S. Harmon, B. White, O. P. Smith, P. V. Jenkins, B. Dahlback, and J. S. O'Donnell Platelet Factor 4 Impairs the Anticoagulant Activity of Activated Protein C J. Biol. Chem., February 27, 2009; 284(9): 5869 - 5875. [Abstract] [Full Text] [PDF]
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J. J. Briede, G. Tans, G. M. Willems, H. C. Hemker, and T. Lindhout Regulation of Platelet Factor Va-dependent Thrombin Generation by Activated Protein C at the Surface of Collagen-adherent Platelets J. Biol. Chem., March 2, 2001; 276(10): 7164 - 7168. [Abstract] [Full Text] [PDF]
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TOP
ABSTRACT
INTRODUCTION
) and in the absence of APC (
).
Error bars indicate standard error of the mean. In (A), the
effect of freeze-fracturing is shown. In the absence of APC, the APTT
was constant, but in the presence of APC, the APTT was reduced even at
a platelet count of 2 × 109/L. In the example
shown, the APC sensitivity ratio of PPP and fresh PRP was 3.2. This was
reduced in freeze-fractured PRP to a ratio of 1.7 at a platelet count
of 400 × 109/L. After ultracentrifugation at
77,000g for 1 hour, the APC resistance phenotype was abolished
(
). In (B, C, and D), the effect of platelet activation with
different agonists is shown: (B) TRAP (n = 3); (C) collagen (n = 7); and (D) A23187 (n = 3).
) and without (
) GR144053F (F = 25.743;
P < .001; ANOVA pairwise comparison).
IIb
3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity and function.
J Biol Chem
266:17106, 1991